Separation of macromolecules (proteins, DNA and RNA) and larger aggregates, such as viruses and cells, has been and continues to be one of the primary objectives in biochemical research. Perhaps the oldest and still most widely used separation technique is solution gradient density centrifugation comprising three basic steps: forming a solution gradient, e.g., of sucrose in a tube; centrifuging a sample into the gradient; and recovering the now-separated samples from various positions in the gradient-containing tube, sometimes referred to as fractionation.
The term "gradient" implies a continuous variation in concentration from top to bottom, e.g., 5% to 45% sucrose. The gradient performs two critical functions. First and foremost, the gradient prevents mixing in a vertical direction. During acceleration and deceleration of the tube in the centrifuge, a mild degree of mixing is induced which, if unchecked by the gradient, would thoroughly mix the contents in the tube. The gradient, however, prevents such mixing of the density differential between adjacent layers. Secondly, heavier sucrose solutions are much more viscous than light sucrose solutions and consequently, there is established a viscosity gradient. Such a viscosity gradient is useful because "g" forces are greatest at the bottom of the tube (highest radius from the center of rotation) and the increased viscosity effectively cancels the increased "g" forces giving a nearly uniform rate of molecule or particle migration from top to bottom, and consequently one can predict the position of the desired molecules at the end of a run.
One of the most serious problems in the constructions of sucrose gradients is reproducibility. It is apparent that the rate of migration of any molecular species through a gradient is subject to the cumulative effects of buoyancy and viscosity of the gradient. Since these two parameters are caused by the shape of the sucrose gradient itself, tube to tube variation in the gradient will produce tube to tube variation in the final position of any molecular species. Often, it is desired to determine whether subtle changes have occurred in the size or shape of cell components, and with very reproducible gradients, such differences may be detected. By the same token, the absolute shape of the gradient is less important so long as the gradient is reproducible.
There has been a steady but slow evolution in the techniques used to form sucrose gradients, beginning with the laborious manual layering of one solution after another into a tube requiring a plurality of pipettes, a steady hand and mountains of patience and time. Such a technique was quickly supplanted with a technique similar to chromatographic technology wherein two solutions, in this case, the highest and lowest sucrose concentrations in a desired gradient, are measured into two adjacent chambers. The mixing chamber (heavy sucrose) is connected to a centrifuge tube on one side and other chamber (light sucrose), on the other side. As the mixing chamber's contents empty into the centrifuge tube, the contents of the other chamber enter and gradually lower their sucrose concentration. As the chambers empty, the outflow approaches the light chamber's concentration. Such chromatography-like technology is the most commonly used technique and produces either linear or exponential gradients with minor modification, but has two major drawbacks, i.e., time and reproducibility. When more than one gradient is desired the outflow must be partitioned, and nothing has yet been developed that will ensure exactly the same flow into each tube. Consequently, a user must watch the level in each tube, clamping off the fast ones until the slow ones catch up, etc. Additionally, there will be slight differences between the gradient in the various tubes because of constant flow adjustments.
Another technique currently in use is a freeze-thaw method, wherein a homogenous solution is introduced into a centrifuge tube and the tube is subjected to a plurality of freeze-thaw cycles. Such a freeze-thaw method suffers from a serious drawback in that, while the freezing and thawing produces a gradient (ice floats and excludes solute molecules from the pure water matrix), any buffer is subjected to the same forces and also ends up as a gradient, producing numerous potential artifacts. Reproducibility is poor because no two tubes thaw out exactly the same way, and also because the gradients decay with time.
Blotting is the generic name for a variety of techniques that permit the visualization of proteins and nucleic acids (DNA & RNA) on filters. Most frequently, these molecules have been separated on a gel made of acrylamide or agarose and have been resolved into a series of "bands" that reflect the molecular weight of each resolved species. These bands resemble the pattern of railroad ties along a track, and in fact, the lanes of bands resolved from a single sample are often referred to as tracks.
These separations are normally carried out under the influence of an electrical field, with the molecules being driven through the molecular matrix by their net electrical charge.
Once the separation is complete, there must be some means of visualizing the bands, since proteins and nucleic acids are not normally visible to the naked eye. Often the gel itself is stained and destained using a dye that is taken up by the molecule in question. Alternatively, the molecules can be made radioactive and their presence detected by exposing the gel to a piece of X-ray film in a process called autoradiography. If more sensitive or specific staining is required, then blotting is the method of choice.
Blotting begins with the electrophoretic or capillary transfer of the bands to the surface of a special piece of filter paper that has a strong affinity for the type of molecule in question. The gel, in the form of a thin slab, and the filter paper are mated in sandwich fashion and the elution of bands onto the filter is carried out. As the bands leave the gel, they adhere to the surface of the filter in precisely the same position they occupied in the gel.
There are two basic classes of methods of detection once bands have been transferred to the filter. In the first class, staining or autoradiography is used to reveal the position of the bands. These methods do not reveal anything new about the molecules and are carried out for a variety of reasons, including increased sensitivity during autoradiography and recovery of purified proteins from stained blots.
The second method involves the detection of bands using a variety of probes which are specific to a fraction of the molecules that has been separated, rather than to all of them. For example, a Western blot is used to detect the AIDS virus. The patient's serum is used to probe a blot that has all the viral proteins separated on it. All serum contains large amounts of proteins called antibodies that are used by the immune system to fight infection. If the person is infected by the AIDS virus, they will have antibodies in their serum that will bind to the viral proteins on the filter. If they are not infected, they will lack such antibodies. The second step is to detect where the antibodies have bound to the blot.
For nucleic acids, a Northern or Southern blot is used. A labelled nucleic acid probe will form a base pair with nucleic acid fragments that have been separated on the blot if and only if they have a sequence which is complementary to that of the probe. The label carried by the probe is then used to reveal the position of those band(s) which are complementary to it.
Thus all three methods, Western, Northern and Southern blots, begin with a filter and a probe. For the blot to be effective, the probe is in solution and must be evenly distributed over the surface of the blot and mixed periodically to ensure maximum sensitivity.
There have been two techniques commonly used to accomplish this; open trays and seal-a-meal bags. The former has been used more with Westerns, which occur at room temperature, while Northerns and Southerns are done in bags because of the high evaporation rates produced by the high temperatures required for the hybridization.
The types of probes used in protein and nucleic acid blot development have been different until very recently. The hybridizations used with Northerns and Southers are relatively straightforward. The filter is treated to remove non-specific binding sites and the probe is added to an overnight, high temperature hybridization. Unbound probe is rinsed off and the blot is exposed by autoradiography.
Westerns are more involved because the presence of the bound primary antibody must be identified. Protein stains are of no use because they will stain non-target proteins as well. Thus a second antibody linked to a special enzyme is incubated with the blot. It is specific for the first antibody and binds only where it finds it bound to target protein. After extensive rinsing to remove all unbound antibody, a colourless substrate is added to the blot. Wherever the enzyme bound to the second antibody exists, the substrate is cleaved and converted to an insoluble coloured product which deposits on the blot and reveals the position of the bands detected by the first antibody. This is a multi-step process that is difficult to carry out in bags because of all the opening and resealing required.
Interestingly, a new technique has recently emerged called chemiluminescence in which the enzyme cleaves a molecule that gives off tiny bursts of light (photons). Thus if a piece of autoradiography film is placed over the filter, the position of bands detected by the probe is revealed as a dark band on the film. This technique has been adapted for non-radioactive detection of nucleic acids in Northerns and Southerns and requires as many steps as the Western technique.